| Infections with influenza virus have a major impact on human health and the economy and cause a high degree of morbidity and mortality annually. Despite the improvement in antiviral therapy during the last decade, vaccination is still the most effective method of prophylaxis and treatment.However, the design of broad-spectrum vaccine target on the “Head” domain becomes very difficult due to the high mutation of HA1. The research shows that the influenza virus HA2 is relatively conservative and the changes of the structure play an important role in the progress of infection. It has become the target of many broadly neutralizing antibodies and is the research hotspots of universal influenza vaccine.The proteolytic activation of HA on the surface of the host cells, from the precursor form HA0 to the mature form HA1 and HA2, is critical for fusion activity. HA1 combines with the receptor of host cells, the virus is taken into cells by endocytosis and at the low p H of endosomes, between p H5 and p H6, a series of conformational change happen to HA2. Some epitopes hidden in the state of pre-fusion will expose for a short time and recognized by the immune system to induce immune response. However, the immune response is very weak due to the short exposure. The design of epitope that can induce a strong immune according to the conserved region of HA2 is the focus of this research.The research shows that No V P domain has three protruding loops. We can insert exogenous antigens into them to present antigens. No V P recombinant protein expressed in E.coli can spontaneously form P-particle which is a 24-copy nanoparticle with a diameter of 20 nm to enhance immunogenicity.The sequences of HA of 17524 H1 10763H3 and 4022 B were firstly analyzed to study conservative rate of HA2, three epitopes including H1:DIWTYNAELLVLLENE H3:DLWSYNAELLVALENQ B:TISSQIELAVLLSNEC that may induce a strong immune response were screened according to the epitope-predicting website. Then we insert three candidate epitopes separately and together into three loops of P protein with a linker(GGGGS) between the epitope and the loop to expose epitopes outside the P-particle when expressed in E.coli and get four chimeric influenza epitope No V P-particle: PP-H1 PP-H3 PP-B and PP-H1-H3-B.We expect to get universal influenza vaccine target on HA2 after some structural characterization including size analysis, TEM observation and immunization of recombinant protein.BALB/c femal mice were immunized with the PP recombinant protein four times with a two-week interval to evaluate immunogenicity of recombinant protein The results show that the titer of H1, H3, B antigen specific antibody of IAV HA was 1.25*105,1.17*105 and 7.03*104. The antibody titer of PP-H1-H3-B recombinant protein immune sera against H1, H3, B epitope was 1.15*105, 9.91*104 and 2.25*104.The cross-binding activity showed that one immune sera can bind the the other two epitopes. The antibody titer of PP-H1 immune sera against H3 and B epitopes was 1.11*105 and 3.14*103. The antibody titer of PP-H3 immune sera against H1 and B epitopes was 1.16*105 and 3.26*103. The antibody titer of PP-B immune sera against H1 and 3 epitopes was 1.59*104 and 1.32*104. This kind of cross-binding provided an evidence that we may get some universal influenza epitopes.Because the epitope is located in HA2, immune serum does not have the activity of HAI in theory. This is consistent with the experimental results of HAI.The antibody typing test showed that Ig M and Ig G1 in immune sera were significantly improved after immunization. So the immune response is mainly humoral immunity and cellular immunity is very weak.Microneutralization test in vitro showed that all the four kinds of sera can neutralize two H3 subtype virus.The ID50 of PP-H1 group against the two H3 subtype virus was 614.0 and 1113. The ID50 of PP-H3 group against the two H3 subtype virus was 908.2 and 1592.8. The ID50 of PP-B group against the two H3 subtype virus was 410.9 and 653.2. The ID50 of PP-H1-H3-B group against the two H3 subtype virus was 1438.2 and 523.2.Virus challenge was performed after immunization of PP-H1-H3-B recombinant protein. After three days, lung tissue was harvested to detect virus copy number. Virus amounts in lungs of mice was 10 3.774-3.797 copies in the PBS control group and 103.153-3.165 copies in the PP-H1-H3-B group. There are significant differences between them. PP-H1-H3-B immune sera can inhibit virus replication effectively in the lungs of mice and reduced viral load.In summary, PP chimeric immunogen is mainly designed according to the characteristics of conformational change of HA2 in the process of virus infection and conserved characteristics of HA2.The immune sera can cross-bind the H1 H3 B epitopes.The PP-H3 and PP-H1-H3-B immune sera have neutralizing activity against H3 subtype virus. This study provides a basis for the development of universal influenza vaccine.However, cellular and humoral immunity should be considered when we are designing virus immunogen. We should balance of the proportion of the cellular and humoral immunity response. Not only the research cost can be reduced, but also the development cycle can be shorten in the application of influenza virus vaccine.Then we can make an effective response to the influenza epidemic. |
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